Congratulations to Luke Piszkin (now a PhD student in the Biophysics Department at the University of Notre Dame) for the first paper in the lab to be first-authored by an undergraduate! Luke’s paper is titled Extremophile enzyme optimization for low temperature and high salinity are fundamentally incompatible and appears in the journal Extremophiles. In the paper Luke explores the molecular basis underlying the intriguing observation that there appear to be very few (no?) extreme halophiles that are also extreme psychrophiles, despite the fact that there are many environments on Earth that are both cold and salty.
One of these environments is Deep Lake, Antarctica, which supports a microbial community dominated by the mesophilic archaeon Halorubrum lacusprofundi (optimal growth temperature of 36 °C). That’s rather surprising given that your typical true psychrophile conks out at about 18 °C. Like all haloarchaea, what H. lacusprofundi can do is tolerate high levels of salt, up to 4.5 M NaCl or 262 g L-1. That level of salt tolerance is not seen among the documented true psychrophiles. Why not?
In the manuscript we posit that it comes down to the different amino acid substitutions needed to adapt a protein to high salt or low temperature conditions. High salt proteins typically have low isoelectric points, derived from more acidic amino acids. The practical implication of this is that they have a more negatively charged surface that requires a high concentration of salt for stability. This is a requirement for the “salt-in” strategists that dominate the most saline environments (such as salt crystallizer ponds). These microbes are primarily archaea but include a few bacteria, and deal with the high salinity of their environment by accumulating high intracellular concentrations of the salt KCl. This maintains their osmotic balance while excluding more harmful salts, but requires proteins that are compatible with high concentrations of KCl. By contrast most halotolerant bacteria (including psychrophiles that inhabit moderate salinity environments) are “salt-out” strategists that accumulate organic solutes to maintain osmotic balance. These solutes impose no particular requirements on intracellular proteins.
The trick is that amino acid substitutions that lead to a lower isoelectric point also decrease the flexibility of the protein. Increased flexibility is the key protein adaptation to low temperature. Thus the fundamental incompatibility between optimization to low temperature and high salinity. To test this idea Luke dusted off a model, the Protein Evolution Parameter Calculator (PEPC), that I developed many years ago in the waning days of my PhD. After updating the code from Python 2 to Python 3 and making some other improvements, Luke devised an experiment to “evolve” core haloarchaea orthologous group (tucHOG) proteins from H. lacusprofundi and the related mesophile Halorubrum salinarum. By telling the model to select for increased flexibility or decreased isoelectric point he could identify how improvements in one parameter impacted the other. As expected, likely amino acid substitutions (based on position in the protein and the BLOSUM80 substitution matrix) that increased flexibility also strongly favored an increased isoelectric point.
Fantastic summary, thanks Jeff!